Aim To investigate the effects of ClC-3 antisense oligonucleotide on H2_O2-induced apoptosis in rat aortic vascular smooth muscle cells. Methods Western-blot was performed to detect the expression of ClC-3 protein. Morphological determination, DNA agarose gel electrophoresis, MTT assay and flow cytometry analysis were performed to measure the morphological changes, DNA ladder, viability and apoptotic rate and effects of ClC-3 antisense oligonucleotide on rat aortic smooth muscle cells. Results The antisense oligonucleotide specific to ClC-3 mRNA decreased the expression of ClC-3 protein and enhanced the apoptotic events induced by H₂O₂ including ultrastructural alteration and DNA ladder. The apoptotic cell death rate increased from 52.8%±13.6% to 75.7%±5.8% (n=6,p<0.01),and whereas the viability further decreased from 48.9%±4.3% to 31.3%±4.3 % (n=6,p<0.01) by ClC-3 antisense oligonucleotide transfection. Conclusion ClC-3 antisense oligonucleotide potentiated apoptosis induced by H₂O₂ in rat aortic smooth muscle cells.